Red blood cell products and the storage of red blood cells in containers free of phthalate plasticizer

ABSTRACT

Red blood cell products and compositions are disclosed. The product includes a container made from PVC or a non-PVC material that is substantially free of a phthalate plasticizer but otherwise includes one or more non-phthalate plasticizers or extractable agents. The product includes a RBC concentrate which has been combined with an additive solution for storing the RBCs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional application of U.S. patent application Ser. No.15/974,914, filed May 9, 2018, now U.S. Pat. No. 11,000,551, which is adivisional of Ser. No. 14/122,847, filed Nov. 27, 2013, now U.S. Pat.No. 9,993,389, which is a U.S. National Stage of PCT Patent ApplicationNo. PCT/US12/56100, filed Sep. 19, 2012 which claims the benefit of U.S.Provisional Patent Application No. 61/536,370, filed Sep. 19, 2011, U.S.Provisional Patent Application No. 61/549,562, filed Oct. 20, 2011, U.S.Provisional Patent Application No. 61/566,409, filed Dec. 2, 2011, andU.S. Provisional Patent Application No. 61/595,891, filed Feb. 7, 2012,all of which are incorporated herein by reference in their entireties.

BACKGROUND

Red blood cells are often separated from whole blood and collected forlater transfusion to a patient in need of red blood cells. For example,red blood cells (hereinafter “RBCs”) may be administered to a patientsuffering from a loss of blood due to trauma, as a post-chemotherapytreatment, or as part of a treatment of one or more blood bornediseases, such as certain anemias and the like. Unless administeredimmediately after collection from a donor, RBCs must typically be storedfor some period of time prior to transfusion. The storage period may beanywhere from a few days to several weeks.

Prolonged storage of RBCs can (negatively) affect RBC function. In orderfor the RBCs to be suitable for transfusion to the recipient, RBCs mustmaintain adequate cell function and metabolism. For example, RBCs mustmaintain an adequate concentration of adenosine triphosphate (ATP) and2,3-DPG. In addition, the presence of lactate must not be too high inthe stored RBCs. Still further, stored RBCs must have acceptably lowlevels of hemolysis. Typically, an acceptable level of hemolysis isbelow 1.0% (in, for example, the U.S.) and 0.8% (in Europe) after 42 daystorage.

Media for providing a storage environment for RBCs that will allow cellfunction and cell metabolism to be preserved and maintained have beendeveloped and are commonly used. The media developed for RBCs canprolong the storage life of RBCs for up to 42 days. Such media (or“storage solutions”) often include a nutrient for the RBCs, a buffer tohelp maintain the pH of the RBCs, electrolytes, a RBCmembrane-protecting compound and other additives to enhance and extendthe life of the RBCs. Examples of widely used and accepted storage mediaare Adsol and SAG-M, available from Fenwal, Inc., of Lake Zurich, Ill.Adsol and SAG-M include sodium chloride, glucose, mannitol, and adenine.Both Adsol and SAG-M have a pH of about 5.0 (referred to herein as “lowpH”) and are substantially isotonic.

Other additive solutions are disclosed in U.S. Patent ApplicationPublication Nos. 2009/0239208 and 2011/0117647 both of which areincorporated by reference herein in their entireties. The additivesolutions disclosed therein are, hypotonic, synthetic aqueous storagesolutions for the prolonged storage of RBCs. The storage media disclosedtherein typically include adenine, mannitol, sodium citrate, sodiumphosphate, and glucose as the nutrient. These hypotonic aqueous additivesolutions have a “high” pH of at least about 8.0.

During storage, concentrated RBCs and the additive solutions in whichthey are stored are typically kept in a sealed container, usually madeof a plastic material. Most typically, the containers approved for thecollection of whole blood and the storage of RBCs are made of apolyvinyl chloride (PVC). Inasmuch as polyvinyl chloride can be somewhatrigid or brittle, a plasticizer is typically incorporated into the PVC.Examples of currently known and used plasticizers for medical grade PVCare DEHP, TEHTM, and the family of citrate esters described in U.S. Pat.No. 5,026,347, the contents of which is also incorporated by referenceherein.

As reported in U.S. Pat. No. 5,026,347 and other literature, such asRock, et al. “Incorporation of plasticizer into red cells duringstorage,” Transfusion, 1984; Horowitz et al. “Stabilization of RBCs bythe Plasticizer, Di(ethylhexyl)phthalate,” Vox Sarquinis, 1985, certainplasticizers may have a beneficial effect on the storage life of RBCs.More particularly, plasticizers such as DEHP and the family of citrateesters have been found to suppress hemolysis of RBCs stored incontainers that include such leachable plasticizers. RBCs stored incontainers made of plasticized PVC or a non-PVC container withplasticizer added (as described in U.S. Pat. No. 5,026,347) havetraditionally been combined with an isotonic, low pH storage solution(such as Adsol). While DEHP plasticized containers have worked well forthe storage of red cells, the use of other container materials andadditive solutions to provide a suitable storage environment for redblood cells remains a topic of keen interest.

Thus, it would be desirable to provide a storage environment for RBCswherein (1) the container is made of (a) a non-PVC material that is atleast substantially free of any leachable phthalate plasticizer or (b)PVC material plasticized with a non-phthalate plasticizers and (2) asuitable storage media. As used herein, the term “storage environment”refers to the materials and solutions that contact the RBCs duringstorage.

SUMMARY

In one aspect, the present disclosure is directed to RBC products. Theproducts include a container in which the wall of the container definesan interior chamber. At least a portion of the wall is made of a plasticcombined with at least one non-phthalate plasticizer or otherextractable agent. The product further includes a suspension of RBCscontained within the chamber. The suspension includes concentrated RBCsin a hypotonic solution. The hypotonic, chloride-free solution includesat least a nutrient, a buffer, and has a pH of greater thanapproximately 8.0. In a more specific aspect, the plastic may be PVC ora non-PVC composition.

In another aspect, the present disclosure is directed to a transfusibleRBC composition. The composition includes a suspension of RBCs thatincludes concentrated RBCs substantially free of a phthalate plasticizerand having a hemolysis level of below at least 1.0% for its storagelife.

In another aspect, the present disclosure is directed to a method forproviding a transfusible red blood cell product. The method includesderiving red blood cell concentrate from whole blood, combining theconcentrate with a hypotonic solution and storing the combination ofconcentrate and solution in a container that includes a polymericmaterial that is substantially free of phthalate.

In a further aspect, the present disclosure is directed to a containerfor storing red blood cell compositions. The container includes one ormore container walls defining an interior chamber. The container wall(s)is made of a polymeric material that is free of phthalate and includesat least a first extractable agent and a second extractable agentwherein each of the at least first and second extractable agents iseffective in suppressing hemolysis in red blood cells. In a morespecific aspect, the polymeric material may include polyvinyl chloride(PVC) or may be a non-PVC material.

In another aspect, the present disclosure is directed to a red bloodcell product that includes a container having one or more containerwall(s) defining an interior chamber. The container wall is made of apolymeric material that is free of phthalate and includes at least afirst and second extractable agent, each of which is effective insuppressing hemolysis in red blood cells. A suspension of red bloodcells is contained within the interior chamber and includes concentratedred blood cells and an additive solution that includes a nutrient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plan view of a typical RBC storage container used forstoring the RBC suspension described herein;

FIG. 2 is a side view of the container of FIG. 1 ;

FIG. 3 is a graph comparing the levels of hemolysis in variouscontainers having different amounts of a plasticizer (including noplasticizer);

FIG. 4 is a graph showing the levels of ATP over a 42-day storage periodof RBCs stored in containers having varying levels of plasticizer(including no plasticizer);

FIG. 5 is a graph showing the levels of 2,3-DPG over a 42-day storageperiod of RBCs stored in containers having varying levels of plasticizer(including no plasticizer);

FIG. 6 is a graph showing the levels of lactate over a 42-day storageperiod of RBCs stored in containers having varying degrees ofplasticizer (including no plasticizer);

FIG. 7 is a graph showing the levels of hemolysis over a 42-day storageperiod of RBCs stored in containers made of a non-PVC polyolefin withoutany plasticizer and including a non-phthalate extractable agent;

FIG. 8 is a graph showing the levels of ATP over a 42-day storage periodof RBCs stored in containers made of non-PVC polyolefin without anyplasticizer and including a non-phthalate extractable agent; and

FIG. 9 is a graph showing the levels of 2,3-DPG over a storage period ofRBCs stored in containers made of non-PVC polyolefin without anyplasticizer and including a non-phthalate extractable agent.

DETAILED DESCRIPTION

Disclosed herein are RBC products that include (a) a RBC composition,and (b) a container for holding the composition during a period ofstorage. The RBC composition itself includes concentrated RBCs that havebeen combined with an additive solution selected to maintain cellfunction and metabolism of the RBCs during prolonged storage (e.g., atleast about 42 days and possibly even up to at least 49 and/or 56 days).The container is typically made of a plastic material and morespecifically a plastic material that does not include a phthalateplasticizer but may otherwise include one or more non-phthalateplasticizers or extractable agents. The RBCs of the RBC product aresuitable for transfusion to a patient.

As noted above, RBC compositions include RBC concentrate and an additivesolution. Concentrated RBCs are derived from whole blood either bymanual or automated separation collection techniques which will be knownto those skilled in the art. RBC concentrates may include some residualamount of plasma. In one embodiment, the RBC concentrate may have mostof its plasma removed as described, for example, in InternationalApplication Publication WO/2011/049709, incorporated herein byreference.

Regardless of how much plasma remains with the RBCs, the additivesolution is one that allows for the extended storage of RBCs (in thecontainers described herein) for over 21 days, over 35 days, up to atleast 42 days, and even up to at least 49 and/or 56 days. In oneembodiment, suitable additive solutions include at least sodiumchloride, glucose (nutrient), mannitol and adenine. In a specificexample, the additive solution includes approximately 111 mM glucose(dextrose), 154 mM sodium chloride, 41 mM mannitol and 2.0 mM adenine.The solutions may have a pH of about 5.0 and are substantially isotonic.Such solution is commonly known as Adsol and is available from Fenwal,Inc., of Lake Zurich, Ill. In another embodiment, additive solutionssuitable for the storage of RBCs in accordance with the presentdisclosure are generally hypotonic and typically (but not necessarily)do not include sodium chloride. Such storage solutions also include anutrient, a buffer, other additives such as sodium citrate, andtypically have a pH of about 8.0 or higher.

More specific examples of hypotonic, high pH additive solutions aredescribed in U.S. Patent Publication Nos. US 2009/0239208 and US2011/0117647, both of which are also incorporated herein by reference.In a specific embodiment, the additive solutions include between about 1to 2.2 mM of adenine; about 20 mM to about 110 mM of mannitol; about 2.2mM to about 40 mM sodium citrate; about 16 mM to about 30 mM sodiumphosphate dibasic and about 20 mM to about 140 mM of glucose. The pH ofthe additive solution is above about 8.0.

In a more specific example, the additive solution useful in the storageof concentrated RBCs in accordance with the present disclosure includesabout 2.0 mM of adenine; about 41 mM of mannitol; or about 25 mM ofsodium citrate; about 20 mM of sodium phosphate dibasic and about 111 mMof glucose. The solution described above is referred to herein as “E-Sol5.”

Thus, concentrated RBCs with some or most of the plasma removed arecombined with additive solutions of the type described above to providethe RBC composition. In one embodiment, the RBC composition includesbetween about 80 to 150 ml of the additive solution combined with about180 to 250 ml of the concentrated RBCs. More preferably, the volume ofadditive solution may be about 100-110 ml.

In the collection of RBCs, it is typical to remove leukocytes from, orat least reduce the number of leukocytes in, the RBCs prior to theirstorage and transfusion. RBCs suspended in an additive solution areoften subjected to a leuko-reduction step which commonly includesfiltration of the RBC/additive solution.

In accordance with one embodiment of the methods and systems disclosedherein, RBCs are subjected to a filtration step or other treatmentwhereby one or both of leukocytes and prions are substantially removed(or the populations of leukocytes and/or prions are substantiallyreduced) from the RBCs. In such embodiment, concentrated RBCs may becombined with an additive solution of the type described above and thecombined concentrated RBC/additive solution composition may be subjectedto the leukocyte and/or prion removal (e.g., filtration) step.

In another embodiment, the RBC concentrate may be “leuko-reduced” and/or“prion-reduced” prior to combination with the additive solution. Thus,in this embodiment, RBC concentrate separated from whole blood isfiltered by passing the RBC concentrate through a leuko-reductionfilter. Alternatively, the whole blood may be subjected toleuko-reduction (i.e., leuko-filtration) and/or prion reduction/removalprior to separation of the RBCs from the whole blood. In any event, theRBCs are “leuko-reduced and/or “prion-reduced.” Filters suitable forremoving leukocytes (and/or prions) from whole blood or RBC concentrate(prior to the addition of the additive solution) include, but are notlimited to, the Sepacell R-500 II, RZ-2000, RS-2000, Flex-Excel,Pure-RC, RZ-200 and R-3000. (Of course, other means for removing and/orreducing the number of leukocytes may also be used.) In this embodiment,the (now) leuko-reduced RBC concentrate is combined with the hypotonicadditive solution of the type described above. The hypotonic additivesolution may be added after introduction of the RBC concentrate into thecontainer, or may already be present in the container at the time of RBCconcentrate introduction.

Leuko-reduced (and/or prion-reduced) RBC concentrate (either with orwithout additive solution) is then introduced into a container which maybe made of (a) a non-PVC material that is at least substantially free ofphthalate plasticizer but preferably includes one or more non-phthalateplasticizer(s) or extractable agent(s), as described in greater detailbelow, (b) a container made of PVC that is at least substantially freeof phthalate plasticizer but is plasticized with or otherwise includesone or more non-phthalate plasticizer(s) or extractable agent(s) (suchas, but not limited to, the above-described DINCH plasticizer), also asdescribed below or (c) any other container suitable for the long termstorage of RBCs.

Containers for storing the RBC compositions disclosed herein may bepermeable to oxygen or at least semi-permeable to oxygen. As shown inFIGS. 1 and 2 , container 10 may include one or more container walls 12which define an interior chamber 15 for receiving the RBC composition20. In one embodiment, two sheets made of a plastic material are broughttogether and sealed along their peripheries 14 to form container 10.Other ways of making container 10 will be known to those of skill in theart and are within the scope of the present disclosure. As shown in FIG.2 , container wall 12 includes an inner surface 13 which contacts theRBCs and an outer surface 17. In one embodiment, container wall may bemade of a single layer of a polymer material, such as PVC or non-PVCpolymer or polymer blend. In another embodiment, container wall 12 maybe made of a multiple sheet laminate wherein inner surface 13 is made ofone material and outer surface 17 is made of a different material.Container 10 may include one or more access ports 16 for connection withtubing 22, docking devices and the like to establish flow into and outfrom the interior chamber 15 of container 10.

In one embodiment, containers useful in the storage of RBCs as describedabove include container walls that are made in whole or at least in partof a plastic material that may include at least one or more polymericcompounds. The one or more plastic and/or polymeric compounds may beblended together and formed into flat sheets that are sealed together inthe manner described above. The polymeric material may be made from orotherwise include polyvinyl chloride (PVC) or one or more non-PVCpolyolefin homopolymers, copolymers or blends thereof. Examples ofsuitable non-PVC polyolefins include polypropylene, polyethylene,including ultra low density polyethylene (ULDPE) and very low densitypolyethylene (VLDPE). Other suitable compounds that may be used in theplastic materials of the containers or as part of the blend for makingthe plastic materials include ethylene vinyl acetate (EVA) and blockcopolymers such as Kraton®. Exemplary formulations and/or thepolyolefins, polyolefin blends or other polymeric compounds which areuseful, either alone or in combination, in the manufacture of containerssuitable for use in the RBC products of the present disclosure aredescribed in U.S. Pat. Nos. 5,026,347, 4,140,162, 5,849,843, and6,579,583, all of which are incorporated herein by reference in theirentireties.

As indicated above, the structure of the container or container wall mayinclude one, two or more layers. The layer formulations may include one,two, three or more components. These structures should be suitable forsterilization by appropriate means, such as steam, ionizing radiation orethylene oxide. Structures suitable for steam sterilization shouldresist distortions to high temperatures up to 121° C. This typicallyrequires incorporation of materials with a melting peak of greater than130° C. The preferred structure of autoclavable material suitable forthe invention will incorporate polypropylene homopolymer or copolymer ata level of 30% or more in at least one of the layers to provide thermalresistance. A suitable polypropylene copolymer is supplied by TotalPetrochemicals (random copolymer 6575). However, thermal resistance canalso be obtained by crosslinking a lower melting material. For example,a 28% vinyl acetate EVA can be crosslinked by ionizing radiationsufficiently to withstand autoclave temperatures even though it has amelting point of 76° C. Suitable materials include Arkema Evatane® 28-03and Celanese Ateva® 2803. The preferred structure is highly flexible,having a composite modulus of not more than 20,000 psi.

In some cases, it may be desirable for the structure to have radiofrequency (RF) response to enable heat sealing. This can be accomplishedby incorporating an RF responsive material such as described in U.S.Pat. No. 5,849,843.

Preferred structures for radiation sterilized applications willincorporate at least 30% of an ethylene based polymer (LDPE, LLDPE,ULDPE, VLDPE, EVA) in at least one of the layers. Structures ofpolypropylene copolymers and polypropylene polymers blended withelastomers such as Kraton® or ULDPE are also suitable for radiationsterilized applications. The preferred structure incorporates lowermodulus components in at least one of the layers to enhance flexibilityand toughness. These lower modulus components can be ultra low densitypolyethylene (ULDPE—typical density less than 0.90 Kg/L), very lowdensity polyethylene (VLDPE—typically density less than 0.925 Kg/L),ethylene vinyl acetate copolymers (EVA) with greater than 16% vinylacetate content, styrene butadiene terpolymers such as Kraton®. ULDPEmaterials are commercially available as Mitsui TAFMER®, Exxon MobilExact® and Dow Affinity®. EVA materials are available as Arkema Evatane®and Celanese Ateva®. These materials are incorporated at levelssufficient to obtain a composite modulus of less than 20,000 psi whilemaintaining resistance to distortion at temperatures greater than 121°C. for autoclaved applications. The disclosure of suitable non-PVCplastics set forth above is not meant to be exhaustive and it will beappreciated that other non-PVC plastics, polymers and blends thereof mayalso be used in the products and compositions of the present disclosure.

Containers of the type described herein may have a container sheet(wall) thickness of between approximately 0.010 to 0.018 inches. Theymay include a non-smooth or any surface finish that minimizes sheetsticking. Typically, containers of the type described herein may have acontainer volume (i.e., interior chamber volume) of approximately 150 mlto 4 L.

As discussed above, containers useful in the methods, systems, andproducts disclosed herein may include PVC or be substantially free ofPVC. Thus, one embodiment, the formulations used to make container walls12 of container 10 are at least substantially free of polyvinylchloride(PVC). At the very least, surface 13 of container wall 12 issubstantially free of PVC. In an embodiment where container 10 is madeof a multiple sheet laminate the sheet providing inner surface 13 may bemade substantially of a non-PVC material while the sheet providing outersurface 17 may be made of a different material. More typically, however,the container wall 12 may be made of a single sheet of a non-PVCpolyolefin, as described above.

Of course, even in containers where the walls 12 are made without anyPVC, some PVC may be present in small amounts. For example, ports 16 mayinclude plasticized PVC. In any event, as used herein, the terms“substantially PVC-free” or “substantially free of PVC” refer tocontainers in which the walls that are in contact with the RBCcomposition, i.e., that part of the container that makes up a part ofthe storage environment, are made from a material that is free of PVC.

Containers suitable for use in the products, systems and methods of thepresent disclosure are at least substantially free of phthalateplasticizer, such as DEHP. This applies to containers where thepolymeric material is PVC plastic as well as where the polymericmaterial is a non-PVC plastic. In the case of a container that includesPVC, such container material will have to be plasticized due to thebrittle nature of PVC. As noted above, the plasticizer is anon-phthalate plasticizer. Non-phthalate plasticizers that may besuitable for use in the PVC containers described above include, forexample, triethylhexyltrimellitate (TEHTM) and the family of citrateesters, as described in U.S. Pat. No. 5,026,347. Preferably, the PVC maybe plasticized with 1,2-cyclohexane dicarboxylic acid diisononyl ester,known by its trade name, DINCH. In an embodiment where the polymer inthe container material formulation is PVC, at least 10%, by weight, ofthe formulation is preferably one or more preferablyhemolysis-suppressing, non-phthalate plasticizers such as DINCH or acitrate ester such as n-butyryltri-n-hexyl citrate. In an embodiment,PVC containers of the type described above may include approximately55-80%, by weight, PVC resin and approximately 20-45%, by weight, ofnon-phthalate plasticizer(s) wherein a preferred plasticizer is DINCH,and less than about 3.0% of stabilizers and lubricants. In a morespecific embodiment, containers of the type described above may includeapproximately 60-70% PVC resin, 20-35% DINCH plasticizer, approximately4-10% epoxidized oil and approximately 0.5-3.0% additionalco-stabilizers and lubricants. DINCH is available from, for example,BASF of Ludwigshafen, Germany.

In the case of non-PVC containers, such containers may likewise be freeof phthalate plasticizers but include non-phthalate plasticizer. Withrespect to either the PVC or the non-PVC containers, at least a portionof the container wall, i.e., the portion or surface that is in contactwith the RBCs during storage, is at least substantially free ofphthalate plasticizer. For example, with reference to FIG. 2 , at leastinner surface 13 (or that portion of inner surface 13 that is in contactwith the RBCs) may be substantially free of phthalate plasticizer. Thus,the storage environment in which the RBCs reside is at leastsubstantially free of phthalate plasticizer. In a more specificembodiment, the storage environment in which the RBCs reside is at leastsubstantially free of phthalate plasticizer, includes a non-phthalateplasticizer and further includes a suitable storage solution (such asthose previously described).

Thus, for example, the PVC or non-PVC container (or more specifically,the container wall) is at least substantially free of a phthalateplasticizer but may include a non-phthalate plasticizer or extractableagent such as the citrate esters described in U.S. Pat. No. 5,026,347,or the DINCH plasticizer described above. Accordingly, suchnon-phthalate plasticizer(s) will be present in and part of the RBCstorage environment. As the containers disclosed herein are often partof a larger processing set that includes tubing, ports, membranes andconnectors in addition to being part of the storage environment, non-PVCand non-phthalate materials of the type described herein may also beused in the manufacture of such other processing set components.

In the embodiments described above, where the plastic container materialmay be PVC or a non-PVC composition, where no phthalate plasticizer oragent is included, and the material includes a single non-phthalateplasticizer (such as DINCH), a hypotonic, high pH storage media maypreferably be used in the red blood cell composition, as demonstrated inStudies 4-6 below.

RBC compositions (which include RBC concentrates and hypotonic, high pHadditive solutions) may be stored in the substantially phthalate-freecontainers. The RBC compositions stored in such containers may be storedfor more than 21 days, more than 35 days and up to at least 42 days oreven up to at least 49 days and/or 56 days, while maintaining acceptablestorage cell function parameters (i.e., a level of ATP, 2,3-DPG,lactate). In particular, RBC compositions stored in the containersdescribed above and that are substantially phthalate-free maintainhemolysis levels below 1.0% and even below 0.8% at, for example, 42 daysof storage. Similarly, the RBC compositions stored for at least about 42days also include ATP, 2,3-DPG, lactate, potassium, phosphate levelsthat are comparable to RBC compositions stored in plasticized PVCcontainers, as shown in the Studies reported herein.

In another embodiment, the plastic composition may include two or moreplasticizers or extractable agents. The plastic container material maybe either PVC or the non-PVC materials described above. Likewise, thestructure of the container may also be as described above (single layeror multiple layers). Thus, in such an embodiment, the plasticcomposition may include a first extractable agent and a secondextractable agent. At least one of the agents is preferably anon-phthalate extractable agent/plasticizer (e.g., not DEHP). In anembodiment where both the first and the second extractableagents/plasticizers are non-phthalate plasticizers, one of theagents/plasticizers may be a non-phthalate, extractable agent that cansuppress hemolysis such as, but not limited to, the citrate estern-butyryl-n-hexyl citrate (BTHC). More preferably, at least the firstand second extractable agents or plasticizers are extractable agents orplasticizers, each effective in suppressing hemolysis in RBCs. Thus, inthe embodiment where BTHC is one of such extractablehemolysis-suppressing agents, the other of the at least first or secondagents or plasticizers may be a non-phthalate plasticizer, such asDINCH, which also is effective in suppressing hemolysis.

In another embodiment, the plastic composition may include a first andsecond extractable agent/plasticizer (wherein one of the first or secondagents/plasticizers is preferably BTHC) and a further or third agent orplasticizer. The further or third agent/plasticizer may likewise be anon-phthalate agent/plasticizer. The third plasticizer may be aplasticizer that is not readily extractable or marginally extractable,such as TEHTM or epoxidized oil (which also acts as a stabilizer).Alternatively or in addition, the third (or further) plasticizer may bemore readily extractable, such as the citrate ester acetyltri-n-butylcitrate (ATBC), which is also effective in suppressing hemolysis, orDINCH. Additional agents or plasticizers may further be included in theformulation of the containers described herein.

In a more specific embodiment, where the polymer in the containermaterial formulation is PVC, at least 10%, by weight, of the formulationincludes preferably two or more non-phthalate plasticizers wherein oneof the plasticizers is DINCH or a citrate ester such asn-butyryltri-n-hexyl citrate (BTHC). In an embodiment, containers of thetype described above may include approximately 55-80%, by weight, PVCresin and approximately 20-45%, by weight, of at least first and secondphthalate plasticizer(s) wherein a preferred plasticizer is BTHC and/orDINCH, and less than about 3.0% of stabilizers and lubricants. In a morespecific embodiment, containers of the type described above may includeapproximately 60-70% PVC resin, 15-30% DINCH plasticizer, 5-15% BTHC,approximately 4-10% epoxidized oil and approximately 0.5-3.0% ofadditional co-stabilizers and lubricants. DINCH is available from, forexample, BASF of Ludwigshafen, Germany.

In another embodiment, the plastic composition may include approximately55%-80%, by weight, PVC and approximately 20%-45% hemolysis-suppressingplasticizer/agent wherein, as a percentage of the overall composition,approximately 3-25% and more preferably 5%-15%, by weight, is a firstplasticizer/agent capable of suppressing hemolysis, such as BTHC. Inaccordance with the present disclosure, the plastic composition mayinclude 55%-80%, by weight, PVC and approximately 20%-45%, by weight, ofcombined hemolysis-suppressing plasticizer/agent wherein, as apercentage of the overall composition, approximately 5-15% is BTHC,2-12% epoxidized oil and 15-30% is one or more of ATBC, DINCH or otherextractable agents, each of which is effective to suppress hemolysis inred blood cells, with approximately 0.5-3.0% of additionalco-stabilizers and lubricants.

The compositions of the present disclosure may also include otheradditives such as anti-blocking and slip agents. Examples of suchanti-blocking and slip agents include derivatives of fatty acid andethylenediamine. More specifically, the agents may be longer chain fattyacids, containing 12 or longer hydrocarbon chains with or withoutmono-unsaturated carbon-carbon bonds, based daiamide withethylendiamine, such as n,n′-ethylene bissteararamide and n,n′-dioleoylethylenediamine. Commercially available compounds of the type describedabove and which may be used in the non-PVC, non-plasticized compositionsof the present disclosure include Acrawax and Glycolube, both availablefrom Lonza of Basel, Switzerland. The anti-blocking and/or slip agentsmay be coated onto the interior surface of the containers or otherwiseincorporated therein.

Compositions that include two or more preferably non-phthalateplasticizers or extractable agents are suitable for storing concentratedRBCs with an additive solution. In such applications, any additivesolution may be used. In one embodiment, the additive solutions may beany known additive solution, such as SAG-M or Adsol (AS-1) availablefrom Fenwal, Inc., of Lake Zurich, Ill. Alternatively, the additivesolution may be the generally hypotonic additive solution describedabove.

In the embodiments where the container walls (or at least the innersurface(s) 13 of the walls) are made of a material completely free ofphthalate, some small trace amounts of phthalate may be present in thecontainer walls as a result of migration from adjoining or adjacentcontainers, from PVC tubing and/or the surrounding environmentgenerally. In addition, as described above, ports 16 may likewiseinclude PVC and as a result may include some plasticizer (includingDEHP). Nonetheless, the presence of some trace amounts of plasticizerattributable to migration from other containers or tubing, or present inthe plastic ports 16, is negligible and such containers are referred toherein as “substantially phthalate-free” or “substantially free ofphthalate.”

Study 1

Whole blood units were collected in CPD anticoagulant in commerciallyavailable blood pack units. Within 15 minutes of collection, the unitswere transferred to non-PVC, non-DEHP polyolefin based containers forpooling. Three units of ABO matched whole blood were pooled together andsplit back into non-DEHP, non-PVC, polyolefin-based containers. Theunits were leukofiltered, centrifuged, and processed into plasma andconcentrated red cells. Approximately 105 ml of E-Sol 5 was added toeach RBC concentrate. The RBC concentrates were then transferred to: (1)a container made of a standard PVC, plasticized with a DEHP (referencedin the Figures as 1.0 DEHP, PVC); (2) a container made of a non-PVC,oxygen semi-permeable material that is a steam sterilizable,multi-component blend that includes a copolymer of polypropylene as itsmajor component with 50% less of the DEHP-plasticizer than the containerin subsection (1) above (referenced in the Figures as 0.5 DEHP, non-PVCA), and (3) a container made of a non-PVC plasticizer-free material thatis a radiation sterilizable, multi-component blend including primarilyethylene vinyl acetate (referenced in the Figures as 0.0 DEHP, non-PVCB).

Units were stored upright for 42 days at 4° C., with weekly sampling forin vitro parameters. On Day 42 DEHP content in the RBC concentrate wasalso measured after thorough mixing of each unit.

Study 2

In a further study, whole blood units were collected into CPDanticoagulant in commercially available PVC, plasticizer blood packunits. Within 15 minutes of collection, whole blood units wereleukofiltered, centrifuged, and processed into plasma and concentratedRBC component. Approximately 105 ml of E-Sol 5 was added to each redcell concentrate, which was transferred to a container made of a semigas-permeable, non-PVC material of the type used in the container ofsubsection (2) in Study 1 described above but substantially free of anyplasticizer (referenced in the Figures as 0.0 DEHP, non-PVC A).

Units were stored upright for 42 days at 4° C., with weekly sampling forin vitro parameters. On Day 42 of storage, DEHP content in the RBCconcentrate was also measured after thorough mixing of each unit.

Study 3

Whole blood units were collected in CPD anticoagulant in commerciallyavailable PVC, plasticizer blood pack units. Within 15 minutes ofcollection, the units were transferred to non-PVC, non-DEHP polyolefinbased containers for pooling. Two units of ABO matched whole blood werepooled together and split back into the original collection containers.The units were leukofiltered, centrifuged, and processed into plasma andconcentrated red cells. Approximately 110 ml of Adsol was added to eachRBC concentrate which was then stored in a container identical to thecontainer used to store red cell concentrate in E-Sol, as describedabove in Study 2 (i.e., referenced in the Figures as 0.0 DEHP, Non PVCA, Adsol and no plasticizer).

Paired units were stored upright for 42 days at 4° C., with weeklysampling for in vitro parameters in one unit from each pair (data shownin FIG. 3 ) and Day 0 and Day 42 sampling only in the other unit fromeach pair (data not shown). On Day 42 of storage, DEHP content in theRBC concentrate was also measured after thorough mixing of each unit.

No major differences were observed in hematocrit, pH, glucose, lactate,phosphate, potassium, 2,3-DPG, and red cell micro particles among thethree arms of Study 1 and the single arm of Study 2 described above. Asshown in FIG. 3 , all E-Sol 5 units passed accepted criteria forhemolysis (below 1.0% and 0.8%, respectively) at Day 42. The Adsol unitsdescribed in Study 3 had 0.8% hemolysis on average on Day 42. ATP,2,3-DPG and lactate levels were also comparable in the E-Sol storedunits, as also shown in FIGS. 4-6 . With reference to Table 1 below,DEHP levels in the non-PVC, non-plasticized containers from Studies 1and 2 showed negligible DEHP levels of 0.7±0.2 and 2.3+0.8 and 0.9 and+0.2 ppm, respectively in the RBC compositions, thereby indicating thatthe RBC compositions that are “substantially plasticizer-free” andstored in E-Sol 5 maintained acceptable hemolysis levels.

TABLE 1 DEHP Study Container/Additive Solution n Content at Day 42 1 1.0DEHP, PVC 9 34.9 ± 4.7  E-Sol 5 1 0.5 DEHP, Non PVC-A 8 16.7 ± 6.4 E-Sol 5 1 0.0 DEHP, Non-PVC-B 10 0.7 ± 0.2 E-Sol 5 2 0.0 DEHP, Non PVC-A9 2.3 ± 0.8 E-Sol 5 3 0.0 DEHP, Non PVC-A 6 1.8 ± 0.5 Adsol

Studies 4 and 5

Paired, ABO matched WB units in CPD were collected into DEHP-PVC bagsand pooled into non-DEHP bags within 15 minutes of collection. Pooled WBunits were split into 2 non-DEHP containers and each unit wasleukofiltered and processed into plasma and a red cell concentrate(RCC). In Study 4, one RCC was stored in a standard DEHP-PVC bag with110 mL Adsol (control) and the other RCC in a DINCH-PVC bag with 110 mLAdsol (test). In Study 5, one RCC was stored in a standard DEHP-PVC bagwith 110 mL Adsol (control) and the other RCC in a DINCH-PVC bag with150 mL E-Sol 5 (test). Units were stored upright at 4° C. for 42 dayswith weekly sampling. On Day 42, DEHP content was quantified for testand control units in Study 1 to ensure there was minimal DEHP exposureduring processing and storage of test units. In vitro storage parameterresults are summarized in Tables 2 and 3 below.

TABLE 2 Study 4 n = 12 pairs Day 1 Day 21 Day 42 ATP Test-DINCH 3.7 ±0.3 4.0 ± 0.5* 2.5 ± 0.4* (μmol/g Hb) Adsol Control- 3.8 ± 0.3 4.3 ±0.6* 2.7 ± 0.6* DEHP Adsol Microparticles Test 10 ± 4  17 ± 5   46 ± 12 (×10³/μL) Control 10 ± 9  16 ± 6   42 ± 9   Hemolysis (%) Test 0.07 ±0.02 0.16 ± 0.05* 0.39 ± 0.20* Control 0.07 ± 0.01 0.12 ± 0.04* 0.25 ±0.11*

TABLE 3 Study 5 n = 6 pairs Day 1 Day 21 Day 42 ATP (μmol/ Test-DINCH4.4 ± 0.3  5.0 ± 0.4* 3.7 ± 0.3* g Hb) E-Sol Control-DEHP 4.4 ± 0.5  4.3± 0.5* 3.3 ± 0.4* Adsol 2,3 DPG Test 13.6 ± 0.6   5.3 ± 3.6* nt (μmol/gHb) Control 12.9 ± 1.4   0.3 ± 0.3* nt Microparticles Test † 4 ± 2  10 ±3*  25 ± 8*  (×10³/μL) Control 5 ± 3  13 ± 2*  33 ± 7*  Hemolysis (%)Test 0.07 ± 0.01* 0.09 ± 0.01* 0.15 ± 0.02* Control 0.06 ± 0.02* 0.09 ±0.01* 0.16 ± 0.01* † normalized for difference in RCC volume *p < 0.05nt = not tested

Study 6

Two (2) ABO matched whole blood units were pooled, split and processedinto concentrated RBCs and plasma within four (4) hours of collection.Approximately 105 ml of E-Sol 5 was added to each. The concentrated RBCsin E-Sol 5 were leukofiltered. Each unit of leukofiltered RBCs in E-Sol5 was stored in a 600 ml plastic container. One container was made of anon-PVC polyolefin without any plasticizer, such as the containerreferenced as 0.0 DEHP, non-PVC B in Table 1 (and of the type disclosedin U.S. Pat. No. 6,579,583). The other container was made of the samenon-PVC polyolefin with DINCH plasticizer coated thereon and gammairradiated. The RBC compositions described above were stored at 4° C.and samples were drawn every 7 days. The results for 2,3-DPG, % ATP andhemolysis are set forth in FIGS. 7-9 .

While the containers, products and compositions disclosed herein havebeen described in connection with various embodiments, it will beapparent to those skilled in the art that modifications and variationsmay be made thereto without departing from the spirit and scope of theinvention.

The invention claimed is:
 1. A method for providing a transfusible redblood cell product substantially free of phthalate plasticizercomprising: (a) deriving red blood cell concentrate from whole blood;(b) combining said concentrated red blood cells with a hypotonicsolution; and (c) storing said combination of concentrated red bloodcells and a hypotonic additive solution in a container that includes apolymeric material that is substantially free of phthalate and includinga first extractable plasticizer comprising 1, 2-cyclohexane dicarboxylicacid diisononyl ester and a second extractable plasticizer comprising acitrate ester.
 2. The method of claim 1 wherein said red blood cellproduct is stored in said container for at least 42 days.
 3. The methodof claim 1 wherein said polymeric material is further combined with aplasticizer comprising triethylhexyltrimellitate (TEHTM).
 4. The methodof claim 1 wherein said polymeric material comprises polyvinyl chloride(PVC).
 5. The method of claim 1 further comprising substantiallyremoving one or both of leukocytes and prions from red blood cells bypassing said red blood cells through a filter.
 6. The method of claim 5comprising removing one or both of leukocytes and prions from said redblood cells by passing said red blood cell concentrate through aleuko-reduction filter.
 7. The method of claim 6 comprising combiningsaid red blood cell concentrate with said hypotonic additive solutionprior to passing said red blood cell concentrate through aleuko-reduction filter.
 8. The method of claim 5 comprising removing oneor both of leukocytes and prions from said red blood cells by passingsaid whole blood prior to deriving said red blood cell concentratethrough a leuko-reduction filter.